Ebselen, Iron Uptake Inhibitor, Alleviates Iron Overload-Induced Senescence-Like Neuronal Cells SH-SY5Y via Suppressing the mTORC1 Signaling Pathway

Increasing evidence highlights that excessive iron accumulation in the brain plays a vital role in neuronal senescence and is implicated in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, the chemical compounds that eliminate an iron overload may provide better protection against oxidative stress conditions that cause the accumulation of senescent cells during brain aging. Ebselen has been identified as a strongly useful compound in the research on redox biology mechanisms. We hypothesized that ebselen could alleviate an iron overload-induced oxidative stress and consequently reverses the senescence-like phenotypes in the neuronal cells. In the present study, SH-SY5Y cells were treated with ferric ammonium citrate (FAC) before ebselen, and the evaluation of the cellular iron homeostasis, the indicators of oxidative stress, and the onset of senescence phenotypes and mechanisms were carried out accordingly. Our findings showed that ebselen ameliorated the FAC-mediated iron overload by decreasing the expression of divalent metal transporter 1 (DMT1) and ferritin light chain (FT-L) proteins. In contrast, it increased the expression of ferroportin 1 (FPN1) protein and its correlation led to a decrease in the expression of the cytosolic labile iron pool (LIP). Furthermore, ebselen significantly reduced reactive oxygen species (ROS) and rescued the mitochondrial membrane potential (ΔΨm). Notably, ebselen restored the biomarkers of cellular senescence by reducing the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and senescence-associated secretory phenotypes (SASP). This also suppressed the expression of p53 protein targeting DNA damage response (DDR)/p21 cyclin-dependent kinase (CDK) inhibitor through a mTORC1 signaling pathway. Potentially, ebselen could be a therapeutic agent for treating brain aging and AD by mitigating iron accumulation and restoring senescence in SH-SY5Y cells.


Introduction
Iron is an essential trace element for normal brain physiological functions such as oxygen transportation, cellular metabolism, myelination, and neurotransmitter synthesis [1].Iron is transported from the circulatory system into the brain parenchymal tissue through the blood-brain barrier (BBB), primarily mediated by either the transferrin/transferrin receptor (Tf/TfR) or the divalent metal transporter 1 (DMT1) pathways.Tese mechanisms predominantly take place within the brain microvascular epithelial cells (BMVECs) [2,3].Neurons acquire iron through DMT1, a key player in a nontransferrin-bound iron (NTBI), which facilitates ferrous iron (Fe 2+ ) uptake from the plasma membrane and its subsequent transportation from endosomes into the cytosolic labile iron pool (LIP).Notably, this process is altered within the context of the aged brain [4].However, the accumulation of excessive iron in the brain can initiate the Fenton reaction, resulting in the generation of hydroxyl free radicals.Given the relatively lower levels of antioxidant defenses, this process can trigger downstream events that eventually lead to cell death [5].
Brain iron overload is the major function of aging and plays an important role in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), although the molecular mechanisms underlying iron-induced neurodegeneration remain an ongoing examination [6].Hence, the maintenance of cellular iron homeostasis must be tightly regulated by a multitude of iron uptake (TfR and DMT1), iron storage (ferritin), iron export (ferroportin 1; FPN1), and its ligand, the master iron-regulatory hormone hepcidin.Tese mechanisms concomitantly occur to ensure the normal physiological levels of cytosolic LIP for the cellular requirements and permit the safe handling of iron in the brain [6][7][8].Recent studies have demonstrated that the increase in the NTBI pool is coupled with the neuroinfammation response to proinfammatory cytokines release and is considered to increase brain aging and several neurodegenerative diseases [9].Te mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Tr protein kinase that belongs to the phosphatidylinositol-3 kinase-(PI3K-) related kinase family.Te mTOR is present as a catalytic subunit in two protein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [10,11].Te mTOR pathway is a central regulator of cellular metabolism and physiology, with important roles in cell growth and proliferation [12,13].Although the involvement of the mTOR pathway in regulating cellular senescence and aging has been studied extensively, the mechanism through which this occurs is still unclear.Furthermore, many of the processes controlled by mTORC1 are dependent on iron as a cofactor and would require regulation of iron homeostasis [14].However, the activation of the mTOR pathway is considered to be the driving force in AD pathogenesis.To date, the mechanism through which the downregulation of mTOR signaling triggers the degradation of amyloid-β and prevents the further accumulation of tau protein has been revealed [15].As a result, targeting the inhibition of mTOR has been widely employed in age-related diseases to slow down the progression of AD [16].
Senescent cells undergo a phenotype development that extends beyond growth inhibition, characterized by irreversible cell cycle arrest with distinct phenotypic alterations and molecular changes within the cells [17].Common features arising in senescent cells are the enhanced enzymatic activity of the lysosomal hydrolase senescenceassociated β-galactosidase (SA-β-gal) and cell cycle checkpoint protein p53, which involve the inhibition of cyclindependent kinases p21 (CDKN1A) activated by DNA damage response (DDR) and is one of the major signaling pathway towards mitochondrial dysfunction [17,18].It is well established that p53 acts as an upstream regulator of mTOR signaling, which highlights the mechanisms underlying the regulation of iron homeostasis and cellular senescence [19].One of the most important activities of senescent cells is developing a complex of senescenceassociated secretory phenotype (SASP).Its prolonged secretion reinforces the spread of premature senescence to neighboring cells and may contribute to the earlier stage of neurodegenerative disease [20].In addition, a strong correlation between senescence phenotypes and AD patient brains has been documented in a transcriptomic analysis study [21].Previous in vitro studies have demonstrated that treating cells with ferric ammonium citrate (FAC) can induce cellular senescence due to iron dysregulation [22,23].Conversely, these senescent cells exhibit resistance to ironinduced ferroptosis [23].Terefore, using synthetic iron inhibitor compounds as novel tools in the treatment of cell senescence and highlighting the efcacy of these agents, are the critical steps for neurodegenerative disease interception.
We speculate that DMT1 iron uptake might be an early target molecule promoting brain iron dyshomeostasis caused by ferric ammonium citrate (FAC).Terefore, the aims of this study were to determine whether ebselen could alleviate and counteract the iron accumulation-induced senescence in SH-SY5Y cells.Te fndings may potentially provide an insight into the therapeutic efect of ebselen to counteract iron burden and cellular senescence via its antiaging efect.

Cell Culture and
Treatments.Human neuroblastoma cell line SH-SY5Y was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; CRL-2266).Cells were cultured in Dulbecco's Modifed Eagle Medium/ Nutrient Mixture F-12 (DMEM/F12) containing 10% heatinactivated fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C in a 5% CO 2 humidifed incubator.Cells were then cultured until the monolayers reached 80% confuence.Te passages from the 3 rd to the 6 th of cells were used for subsequent experiments.Advances in Pharmacological and Pharmaceutical Sciences Once the cells reached approximately 80% confuence, cells were seeded into culture plates.After an overnight serum deprivation, 100 μM FAC in the serum-free medium was applied to the cells for 24 h.Tereafter, the diferent concentrations of ebselen in serum-free medium (5 and 10 μM) were applied for an additional 24 h.Te culture duration for FAC treatment was based on the preliminary time-course experiments, that increased the susceptibility of the expression of iron importer DMT1 protein (see Figures 1(a) and 1(b)).

Cell Viability Assay.
Te cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St. Louis, MO, USA).Briefy, SH-SY5Y cells were seeded into 96-well plates at a density of 2 × 10 4 cells/well.Cells were then treated with 100 μM FAC for 24 h before the addition of the various concentrations of ebselen (0, 3, 5, 10, and 20 μM) and incubated for 24 h.Tese were followed by the inclusion of 2 mg/ml MTT solution in each well and the 2-hour incubation in an incubator.Te MTT solution was then completely removed and dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan crystals.Te amount of MTT formazan product was measured at 570 nm using the Synergy HT microplate reader (BioTek, Winooski, VT, USA).Cell viability was expressed as a percentage relative to the untreated control.

Intracellular Calcein-Chelatable Iron Assay.
Te levels of the cytosolic labile iron pool (LIP) were estimated using the metal-sensitive fuorophore calcein-AM [31] from Invitrogen (Carlsbad, CA, USA) according to the manufacturer's instructions.Briefy, SH-SY5Y cells were seeded into 96-well plates at a density of 2 × 10 4 cells/well.Cells were then treated with 100 μM FAC for 24 h before the addition of ebselen (5 and 10 μM) and incubated for 24 h.Cells were subsequently washed twice with 1X phosphate-bufered saline (PBS) and incubated with 1 μM calcein-AM at 37 °C for 30 min.Te excessive calcein-AM on the cell surface was gently washed twice with 1X PBS followed by the measurement of the fuorescence intensity using the Synergy HT microplate reader (BioTek, Winooski, VT, USA) at excitation/emission wavelengths of 485/538 nm.Te images of the cells were acquired immediately and analyzed by using a fuorescence microscope (ZEISS, Germany).

Measurement of Intracellular Reactive Oxygen Species (ROS).
Te intracellular ROS levels were assessed using the cell-permeable reagent fuorogenic probe, 2′-7′dichlorofuor escin diacetate (DCFDA) from Termo Fisher Scientifc Inc. (Waltham, MA, USA) according to the manufacturer's instructions.Subsequently, the supernatants were completely discarded and the cells were incubated with 1X PBS containing 10 μM DCFDA at 37 °C for 30 min.Te intensity of 2,7-dichlorofuorescein (DCF) fuorescence was measured using the Synergy HT microplate reader (BioTek, Winooski, VT, USA) at excitation/emission wavelengths of 488/ 520 nm.Te data were presented as the percentage of ROS relative to the untreated control.

Measurement of Mitochondrial Membrane Potential (ΔΨm).
Te ΔΨm was measured using tetraethylbenzim idazolyl-carbocyanine iodide (JC-1) as the indicator of mitochondrial damage (Termo Fisher Scientifc Inc., Waltham, MA, USA).Briefy, SH-SY5Y cells were seeded into 96-well plates at a density of 2 × 10 4 cells/well and treated as previously described above.After the treatment, cells were incubated with 5 μM JC-1 at 37 °C for 30 min.Cells were then gently washed twice with 1X PBS and the fuorescence intensity was measured using the Synergy HT microplate reader (BioTek, Winooski, VT, USA).Te wavelengths of excitation/emission for green-fuorescent monomers were set at 490/530 nm and at 525/590 nm for red fuorescent J-aggregates.Te data were presented as the ratio of monomer/J-aggregates.

Senescence-Associated β-Galactosidase Staining (SAβ-Gal).
Te cellular senescence was performed using the senescence β-galactosidase staining kit (Cell Signaling Technology, Inc., Danvers, MA, USA) according to the manufacturer's instructions.In brief, SH-SY5Y cells were seeded into 24-well plates at a density of 3 × 10 4 cells/well and treated as previously described above.Cells were washed twice with 1X PBS and fxed in 4% (v/v) formaldehyde for 10 mins at room temperature.Tis was followed by washing with distilled water for three times and then staining with the β-galactosidase detection solution (pH 6.0) at 37 °C in a non-CO 2 incubator for 16 h.Cells were observed under an Axiocam 305 color, a phase-contrast microscope (ZEISS, Germany) and the bright blue-staining cells were considered as the β-galactosidase positive cells.Cells were eventually counted for three random vision felds and the calculation of the senescence rate was performed.

Measurement of Senescence-Associated Secretory Phenotype (SASP).
Te SASP was quantifed from culture supernatants using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.After the treatment, the particulates were removed by centrifugation at 10,000 × g at 4 °C for 10 min and the supernatants were collected and frozen at −80 °C until further analysis.Te levels of cytokines (IL-6 and IL-8) were assessed using ELISA kits specifc to humans (R&D system).Te absorbance was measured at 450 nm following the manufacturer's reference using the Synergy HT microplate reader (BioTek, Winooski, VT, USA) and the results were expressed in ng/ml.

Statistical Analysis.
All values are presented as mean ± standard error of the mean (SEM) from at least three independent experiments performed in triplicate.Comparison between groups was analyzed by using one-way ANOVA with Bonferroni's tests.Diferences were considered statistically signifcant at P < 0.05.All statistical analysis was performed using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA).

Ebselen Improves SH-SY5Y Cell Viability against FAC-
Induced Cytotoxicity.To determine whether the efective concentration of ebselen against the FAC-induced cytotoxicity and the viability of SH-SY5Y cells exposed solely to various concentrations of ebselen (3, 5, 10, and 20 μM) were measured by the MTT assay.Te viability of the cells treated with ebselen alone compared to the untreated control cells signifcantly decreased at the concentration of 20 μM and promoted cell proliferation at the lowest concentration (3 μM of ebselen) (Figure 2(a)).Terefore, the viability efect of 5 μM and 10 μM ebselen treatments were further investigated after the exposure to the 100 μM FAC for 24 h (Figure 2(b)).Te results showed that ebselen signifcantly improved the reduction of cell viability compared with the FAC-treated cells (Figure 2(c)).Tis highlighted the importance of ebselen in reducing SH-SY5Y cells' toxicity against FAC-induced cytotoxicity.Subsequently, the efective concentrations of FAC (100 μM) and ebselen (5 and 10 μM) were used in all experiments.Figure 1: Ebselen attenuated changes in iron regulation following FAC-induced iron overload in SH-SY5Y cells.Cells were exposed to 100 μM FAC for 0, 6, 12, and 24 h.(a) Western blot analysis of the DMT1 in cells exposed to 100 μM FAC at diferent times and (b) the quantitative analysis of the protein expression normalized using β-actin.Cells were exposed to FAC (100 μM) for 24 h prior to ebselen (5 and 10 μM) for 24 h.(c) Western blot analysis of the DMT1, FPN1, and FT-L, and the quantitative analysis of the (d) DMT1, (e) FPN1, and (f ) FT-L protein expression were normalized using β-actin, respectively.Te data are the mean ± SEM of the three independent experiments performed in triplicate.* P < 0.05 and * * * P < 0.001 vs. the untreated group.* P < 0.05 and * * * P < 0.001vs.the FAC alone group (see supplementary data 1-2 for the original image of Figure 1(a) and supplementary data 3-6 for the original image of Figure 1(c), respectively).FAC (0, 6, 12, and 24 h) was determined in SH-SY5Y cells.Te results showed that the cells treated with FAC for 24 h markedly upregulated DMT1 protein expression compared to the untreated control cells (Figures 1(a) and 1(b)) (see Supplementary data 1-2 for the original image of Figure 1(a)).Terefore, this exposure time (24 h) was selected for the examination of the efect of ebselen in the SH-SY5Y cells after 100 μM FAC treatment.Te results revealed that ebselen signifcantly decreased FAC-induced iron infux into the cell through DMT1 compared with that of the FAC-treated group (Figures 1(c) and 1(d)).Te impact of ebselen on the key components in iron homeostasis in FAC-treated SH-SY5Y cells was also evaluated.FPN1, the only mammalian iron exporter identifed to date [33] and FT-L, the major protein responsible for intracellular iron storage, were measured using Western blot analyses (see Supplementary data 3-6 for the original image of Figure 1(c)).FAC alone signifcantly reduced FPN1, but increased FT-L protein expressions compared with that of the untreated control cells.Tis was rescued following the ebselen treatment (Figures 1(b), 1(e), and 1(f)) which attenuated changes in the iron regulation.Moreover, ebselen also inhibits iron infux that may accelerate the extracellular iron transport.

Ebselen Reduces the Intracellular LIP Level in FAC-
Induced Iron Overload in SH-SY5Y Cells.Te potential effects of ebselen on the cytosolic LIP level in FAC-treated SH-SY5Y cells were evaluated using the calcein-AM assay; and the reduction in calcein-AM fuorescence intensity signifes an increase in the cytosolic-chelatable Fe 2+ [34].Te patches of green fuorescence were notably increased in the cytosol and difused into lysosomes, where they were cleaved by the lysosomal esterase resulting in the calcein-AM penetration (Figure 3(a)).Terefore, the results indicated that the cytosolic LIP level signifcantly decreased following the ebselen treatment.However, this was rescued by the ebselen treatment (Figure 3(b)).Tese fndings suggest that ebselen could directly inhibit iron infux and control the levels of cytosolic LIP in cultured SH-SY5Y cells.

Ebselen Suppresses ROS Accumulation and Rescues the Loss of ΔΨm by FAC-Induced Iron Overload in SH-SY5Y
Cells.It is well documented that excessive iron is involved in an imbalance of redox homeostasis, resulting in the increase of intracellular ROS levels and oxidative stress [35].Tis research investigated the antioxidative property of ebselen against FAC-induced intracellular ROS formation in SH-SY5Y cells using a DCFH-DA probe.Te treatment with FAC alone signifcantly increased the intracellular ROS level compared with the untreated control cells.Interestingly, this was signifcantly decreased by ebselen treatment and the level was comparable to that in the control group (Figure 3(c)).Tese fndings suggest that ebselen possesses antioxidant properties, which could potentially dampen the depolarization of mitochondrial membrane potential (ΔΨM) of aging neuronal cells caused by oxidative stress.Te potential efects of ebselen on the rescuing of the oxidative stress-induced ΔΨm impairment were confrmed using the JC-1 probe.Te results revealed a signifcant decrease in the ratio of green/red fuorescence intensity in the FAC-treated cells, which implied the impairment of ΔΨM.Tis was rescued by the ebselen treatment, resulting in the increase of the green/red fuorescence intensity ratio (Figure 3(d)).

Ebselen Restores SH-SY5Y Senescence via Inhibiting the mTORC1 Signaling Pathway.
Te potential mechanisms of ebselen in restoring the iron overload-associated senescence and oxidative stress in SH-SY5Y cells were evaluated using a collection of cellular senescence markers including SA-β-gal, SASP, and cell cycle inhibitors activated by DNA damage response (DDR).Tis is due to the fact that senescent feature alone is insufcient to determine a senescent state.Te iron overload in the SH-SY5Y cells is frmly associated with an increased number of SA-β-gal positive cells compared with the untreated control cells, which were almost unafected.In contrast, the SA-β-gal positive cells were partially reverted following the ebselen treatment, particularly at a concentration of 10 μM (Figures 4(a   senescence and chronic infammation in the aging brain, the expression of a common SASP component, including IL-6 and IL-8, using ELISA was performed.Te results revealed that there was a marked increase in IL-6 and IL-8 proteins in the senescent SH-SY5Y culture media, which was signifcantly reduced after the ebselen treatment (Figures 4(c) and 4(d)).Furthermore, the iron overload and the overproduction of ROS increased the activation of p53 and the expression of downstream proteins p21, which were efectively reversed by ebselen (Figures 5(a)-5(c)) (see Supplementary data 7-9 for the original image of Figure 5(a)).Te investigation of mTORC1 activity was also carried out and the results showed that the oxidative stress induced the phosphorylation of mTORC1.Conversely, ebselen signifcantly downregulated the phosphorylation of mTORC1 (Figures 5(d)-5(f)), demonstrating that iron overload creates neuronal senescence through the activation of the mTORC1 signaling pathway (see Supplementary data 10-12 for the original image of Figure 5(d)).

Discussion
Iron accumulation is the function of aging as it plays a pivotal role in activating a cascade of pathophysiological processes in cellular senescence via oxidative stress.Ebselen, a restoring antiaging agent, was investigated in the present study to its restoring potential in the iron overload-induced senescence-like neuronal cells, SH-SY5Y.
Iron overload in the brain amplifes neuronal toxicity through its redox-active efects, leading to cellular senescence, which preferentially occurs in physiological brain aging and corresponds to the severity of neurodegenerative diseases, especially AD and PD lesions [36,37].Given the last decade of clinical trials, drug therapeutic for AD has largely been directed at the Aβ peptide, owing to the hypothesis of disease that it is a critical initiator of the progression of lesions [38].However, medication to modify the pathogenesis of AD has not yet been identifed.Ebselen, on the other hand, is a widely recognized synthetic peroxynitrite scavenger compound that mimics the activity of GPx, which catalyzes essential reactions that provide protection against oxidative damage [24].Ebselen has been widely used as a therapeutic agent in the clinical trials of various diseases [39,40].A recent study demonstrated that ebselen acts as a potent anti-infammatory agent by suppressing the TRAF2-ASK1-SEK1 signaling pathway, which is essential for endoplasmic reticulum stress-induced cell death [41], and is consistent with the fact that ebselen has neuropharmacological properties, which possess antioxidant and anti-infammatory activity and free radicals scavenging [42].In addition, dyshomeostasis of brain iron causes aggregation of Aβ peptides and tau protein, consequently promoting neuronal cytotoxicity, which is a pathological hallmark of AD [43,44].Several studies have revealed the potential efects of ebselen in preventing the progression of ADrelated pathology and enhancing the spatial learning and memory in two-month-old 3 × Tg-AD mice, suggesting that ebselen could be a potential therapeutic agent for the prevention or treatment of AD [45].Furthermore, ebselen also has multifunctional properties, including reversing oxidative stress, neuronal apoptosis, and memory impairment in a sporadic model of AD induced by intracerebroventricular administration of streptozocin [27].Tese fndings imply that ebselen could be a therapeutic target for AD.Iron has also been shown to enhance ferroptosis, a type of regulated necrosis characterized by iron-induced lipid peroxidation that causes catastrophic membrane rupture, which is controlled by GPx4, an event that occurs in AD progression [46].Due to the GPX mimetic properties, the selenium compound ebselen could reduce neuronal ferroptosis in traumatic brain injury with cerebral ischemia and intracerebral hemorrhage, respectively [47,48].Nonetheless, there are few studies elucidating the benefts of ebselen as a treatment strategy for curing brain iron overloadassociated senescence-like neuronal cells that accelerate the development of AD.
Te mechanism underlying iron progressively accumulating in senescent cells has not been fully understood.We frst examined the potential of ebselen to alleviate the dysregulation of iron homeostasis in SH-SY5Y cells following the exposure to FAC.Ebselen has been identifed as a DMT1 inhibitor, which plays a vital role in direct DMT1 transport blockage and iron-catalyzed damage limitation [28].Tis could reverse the dopaminergic neuronal loss and motoric defcit in the brain regions implicated in PD [49].Neuronal iron transport is principally dependent on the NTBI transport pathway via DMT1 [4].Our results demonstrated that ebselen signifcantly decreased DMT1 expression upon the increase of intracellular iron level, indicating that the high-afnity property of iron chelator inhibits iron uptake and pumps iron into the cytosol [31,50].In addition, FAC-induced dysregulation of iron homeostasis was considerably mitigated by ebselen.Since ebselen increased FPN1 expression, while decreasing the expression of FT-L and the cytosolic LIP levels; it might exert critical routes to DMT1 inhibitor-mediated FPN1 degradation that plays an essential role in the export of iron.Possibly, ebselen may chelate labile iron to form a relatively stable compound to maintain iron homeostasis [29,30].
Te collective evidence shows that Fe 2+ is an efective modulator to catalyze the conversion of H 2 O 2 to hydroxyl radicals via the Fenton reaction [5].Tis is in line with many studies which have reported that increased LIP leads to a marked increase in ROS formation, the key propagation to DNA damage and mitochondrial function decline observed in mammalian aging [51,52].Our results showed that ebselen markedly decreased intracellular ROS levels and increased ΔΨm following the iron overload in SH-SY5Y cells.Tese outcomes provide a strong evidence supporting the direct property of ebselen as a powerful antioxidant and radical scavenger in the ischemia/reperfusion model [25].As a consequence of the extensive oxidative injury, cells could either react toward a defensive mechanism or the pathological process, leading to mitochondrial dysfunction.Tis is apparently distinct in the accumulation of senescent cells and secretory phenotype [53,54].
Despite being postmitotic cells, neurons could be capable of undergoing senescence in response to oxidative stress [55].Further experiments must be carried out to evaluate Advances in Pharmacological and Pharmaceutical Sciences whether iron accumulation might be a critical factor in brain aging and to assess the property of ebselen on antiaging in reliable neuronal senescence.Te molecular mechanism responsible for cellular senescence demonstrated the downregulation of the nuclear receptor coactivator 4 (NCOA4), a selective cargo receptor for autophagic turnover of ferritin, which induces ferritin accumulation and increases DNA instability and lysosomal dysfunction [56,57].Consequently, senescent cells likely accumulate iron partially due to failure to degrade ferritin [58], thereby sequestering iron in an inactive state and leading to cell-cycle arrest [23].Besides, SA-β-Gal is the most widely used marker of senescence derived from lysosomal enzymes and can be detected at early stages.It is well established that excessive ROS strongly triggers cellular senescence via regulating multiple pathways.Proinfammatory cytokines, termed senescence-associated secretory phenotype (SASP), can increase c-Jun N-terminal kinase (JNK) phosphorylation [59] and activate the pathway [30], thus accelerating cellular senescence.Te SASP paracrine signaling is a crucial pathological feature of cellular senescence that causes local chronic infammation [18,60].Not only glial cells are responsible for releasing proinfammatory cytokines, but also neurons, resulting in a self-propelling cycle of neuroinfammation and eventually neurodegeneration [61].Our results demonstrated that ebselen could restore neuronal senescence by signifcantly reducing β-gal activity and decreasing the expression of IL-6 and IL-8 levels, which is related to its potent anti-infammatory properties [62].
In addition, the appearance of senescence phenotype in our study demonstrated that neurons respond to the damage by activating the p53 pathway and its downstream, p21, known as CDKN1A (cyclin-dependent kinase inhibitor 1) [17,18].Phosphorylation of p53 has been shown to be critical for telomeric stress-induced cellular senescence and is closely linked to DNA double-stranded breaks (DSBs) and DNA damage response (DDR) activation [63].Ebselen notably restored neuronal senescence by the downregulation of p53/ p21 expression.Te results provide evidence that ebselen could decrease the acceleration of senescence through the reduction of ROS levels.By simultaneously investigating the signaling pathway involved in cellular senescence in parallel experiments, we found that ebselen could induce the downregulation of mTORC1 expression, a key mediator of cellular senescence.It has been reported that oxidative stressdependent mitochondrial depletion exacerbates the proaging phenotype thereby promoting the mTOR phosphorylation cascade [64].Terefore, the inhibition of the mTORC1 pathway by ebselen might restore iron overload-induced senescence-like neuronal cells, SH-SY5Y, by suppressing the SASP regulatory proteins, thereby improving the mitochondrial integrity and reversing cell cycle arrest.Advances in Pharmacological and Pharmaceutical Sciences

Conclusions
We conclude that excessive iron plays a pivotal role in accelerating the processes of brain aging, especially neurodegenerative diseases.Our study provides evidence that ebselen, a DMT1 inhibitor, might be one of the therapeutic options for mitigating iron overload-associated senescence-like neuronal cells, SH-SY5Y, through direct and indirect antioxidative and anti-infammatory activities (Figure 6).While we fnd that these studies ofer signifcant insights that contribute to the scientifc community's ongoing interest, it remains crucial to recognize that our current work is merely the initial step.Te efcacy of ebselen appears to prompt the need for subsequent in vivo studies, which we anticipate will be undertaken in the near future.Tese studies will likely pave the way for eventual clinical trials, constituting a critical phase in uncovering the full potential of ebselen as a prospective neurodegenerative medicine.In our perspective, our present research and the forthcoming studies will collectively advance our understanding of ebselen's therapeutic value and contribute to addressing the challenges posed by neurodegenerative diseases.Te combination of in vivo investigations and clinical trials will provide deeper insights into ebselen's efcacy and its potential impact as a future therapeutic medicine.Advances in Pharmacological and Pharmaceutical Sciences

2
Treated SH-SY5Y Cells.Te expression of iron uptake-mediated DMT1 in response to the varied exposure times of 100 μMkDa β

Figure 2 :
Figure 2: Ebselen improved viability in SH-SY5Y after the exposure to FAC.Te cell viability was determined using the MTT assay.(a) Cell viability after the exposure to the serum-free medium containing various concentrations of ebselen for 24 h.(b) Ebselen treatment paradigm following FAC-induced intracellular iron accumulation.(c) Cell viability after the exposure to FAC (100 μM) prior ebselen (5 and 10 μM).Te data are the mean ± SEM of the three independent experiments performed in triplicate.* P < 0.05 and * * P < 0.01vs.the untreated group.

Figure 3 :
Figure 3: Ebselen reduced the intracellular iron level and oxidative stress following FAC-induced iron overload in SH-SY5Y cells.Cells were exposed to FAC (100 μM) for 24 h prior to ebselen (5 and 10 μM) for 24 h.Te intracellular iron level based on the redox-active cytosolic LIP was examined using calcein-AM.(a) Representative images of cytosolic LIP were observed by using fuorescence microscope and taken at ×200 magnifcation.(b) Te quantitative analysis of the intracellular iron level and the decreased calcein-AM fuorescence intensity indicate an increased intracellular iron level.(c) Te quantitative analysis of the intracellular ROS level using DCFH-DA probe.(d) Te quantitative analysis of ΔΨm using JC-1 dye.Te data are the mean ± SEM of four independent experiments performed in triplicate.* * * P < 0.001 vs. the untreated group.* * P < 0.01 and * * * P < 0.001vs.the FAC alone group.

Figure 4 :
Figure4: Ebselen rescued iron overload-induced neuronal senescence.Cells were exposed to FAC (100 μM) for 24 h prior to ebselen (5 and 10 μM) for 24 h and then the senescence markers were measured.(a) Representative images of SH-SY5Y stained with SA-β-gal, the images were obtained by phase-contrast microscopy, and taken at ×200 magnifcation.(b) Te percentage of SA-β-gal-positive cells per feld and three vision felds from each replica were scored.Te amount of SASP secretion, (c) IL-6, and (d) IL-8, was analyzed using ELISA.Te data are the mean ± SEM of the three independent experiments performed in triplicate.* * * P < 0.001 vs. the untreated group.* * P < 0.01 and * * * P < 0.001vs.the FAC alone group.

Figure 5 :
Figure5: Ebselen counteracts neuronal senescence through the mTORC1 pathway.Cells were exposed to FAC (100 μM) for 24 h prior to ebselen (5 and 10 μM) for 24 h and then the expression of markers for the cell cycle was measured.(a) Cell cycle checkpoint protein p53/ cyclin-dependent kinase (CDK) inhibitors p21 were evaluated using the Western blot analysis.Te quantitative analysis of the expression of (b) p53 and (c) p21 was normalized using β-actin, respectively.(d) Western blot analysis of phosphorylated p-mTORC1 and total mTORC1.Te quantitative analysis of the expression of (e) p-mTORC1 and (f ) total mTORC1 was normalized using β-actin, respectively.Te data are the mean ± SEM of the three independent experiments performed in triplicate.* * * P < 0.001 vs. the untreated group.* P < 0.05, * * P < 0.01, and * * * P < 0.001vs.the FAC alone group (see supplementary data 7-9 for the original image of Figure5(a) and supplementary data 10-12 for the original image of Figure5(d), respectively).

Figure 6 :
Figure6: A schematic model representation of the proposed mechanism of ebselen on FAC-induced iron overload associated with senescent-like neuronal cells, SH-SY5Y.FAC facilitates an increased intracellular iron levels via DMT1, the main pathway responsible for cellular iron uptake in neurons.Subsequently, increasing intracellular ROS formation via the Fenton reaction that mediates proinfammatory cytokine release leads to the damage of mitochondrial membrane and DNA.Excessive intracellular iron levels are undoubtedly one of the main causes of cellular senescence, leading to organismal aging.Ebselen is a DMT1 inhibitor that could decrease intracellular iron levels by increasing the expression of FPN1 iron exporter and decreasing FT-L iron storage.Furthermore, ebselen could rescue neuronal senescence by reducing ROS levels and by improving ΔΨm and cellular senescence markers, including SA-β-gal, SASP profle, and p53/p21 via inhibiting the mTORC1 signaling pathway.